The present application is the national stage under 35 U.S.C. 371 of international application PCT/EP98/06815 filed Oct. 28, 1998 which designated the United States, and which international application was published under PCT Article 21(2) in the English language.
This invention relates to a fluorescence detection assembly for determination of relevant vegetation parameters comprising an excitation source consisting in a low power laser device with an excitation wavelength in the red spectral region, a beam forming optical device, a dichroic beam splitter, a basic fluorescence detector system including an entrance optical device receiving fluorescence emission via said dichroic beam splitter and an interference filter blocking out the elastic back scatter signal, an electronic detection device for detecting a fluorescence signal, and an electronic trigger and timing device.
First of all the phenomenon of chlorophyll fluorescence will be discussed now.
The absorbed photosynthetic active radiation (PAR) of the solar irradiation (380 nm less than xcex less than 750 nm) is used by plants primarily to convert the absorbed energy in chemically bound energy (photosynthesis) and stored as chemical energy. This process is directly linked with the uptake of carbon dioxide and the release of oxygen (called primary productivity). Two other pathways are possible for the absorbed energy to keep plants energetically balanced. First, the emission of thermal energy and second, the emission as fluorescence light may be used for regulation.
The thermal energy budget is filled up with solar energy from the visible (VIS) and the short wave infrared (SWIR) range of the solar spectrum. SWIR radiation is directly absorbed by the leaf internal water content. The VIS range contributes via the exciton transfer inside the antenna pigment of the reaction centers (PS I; PS II) and light harvesting complex (LHPC). In this process the absorbed photon energy is transformed to energy quantities required by PS I and PS II. The surplus of energy is stored in oscillating and rotation energy levels and thus finally converted into heat.
At the PS I and PS II the absorbed energy quantities may be used by the so called light reactions, may be transferred to heat or finally emitted as fluorescence light. The emitted fluorescence in the red spectral region is due to the chlorophyll molecules associated to PS I, PS II and the LHPC. The conversion probabilities for heat and fluorescence are considered constant in time, whereas the conversion rate at the light reaction is considered as a function of the state of the reaction center (electron transfer chain) and the phosphorylation state of the photosynthetic active cell membranes. The following Equation (1) will describe the fraction of sun induced chlorophyll fluorescence light (FSun(t)) which is emitted by the reaction centers:                                                         F              Sun                        ⁡                          (              t              )                                =                                                    k                Fluorescence                                                              k                  Fluorescence                                +                                  k                  Heat                                +                                                      k                    Photosynthesis                                    ⁡                                      (                                          Φ                      ,                      M                                        )                                                                        *                                          ∫                PAR                            ⁢                                                I                                      Abs                    -                    Sun                                                  ⁢                                  ⅆ                  λ                                                                    ⁢                  
                ⁢                                                                                                  k                    i                                    ⁢                                      :                                    ⁢                                      xe2x80x83                                    ⁢                  conversion                  ⁢                                      xe2x80x83                                    ⁢                  probability                  ⁢                                      xe2x80x83                                    ⁢                  for                  ⁢                                      xe2x80x83                                    ⁢                  fluorescence                                ,                                  heat                  ⁢                                      xe2x80x83                                    ⁢                  and                  ⁢                                      xe2x80x83                                    ⁢                  photosynthesis                                                                                                                          φ                  ⁡                                      (                    t                    )                                                  ⁢                                  :                                ⁢                                  xe2x80x83                                ⁢                state                ⁢                                  xe2x80x83                                ⁢                of                ⁢                                  xe2x80x83                                ⁢                the                ⁢                                  xe2x80x83                                ⁢                reaction                ⁢                                  xe2x80x83                                ⁢                center                                                                                                          M                  ⁡                                      (                    t                    )                                                  ⁢                                  :                                ⁢                                  xe2x80x83                                ⁢                phosphorylation                ⁢                                  xe2x80x83                                ⁢                of                ⁢                                  xe2x80x83                                ⁢                membrane                                                                                                          I                                      Abs                    -                    Sun                                                  ⁢                                  :                                ⁢                                  xe2x80x83                                ⁢                absorbed                ⁢                                  xe2x80x83                                ⁢                spectral                ⁢                                  xe2x80x83                                ⁢                                  irradiance                  .                                                                                        (        1        )            
From this formula it can be seen that the behaviour of the time dependent chlorophyll fluorescence gives access to the relative changes of the photosynthetic activity if one assumes that xe2x80x9cxcfx86xe2x80x9d and xe2x80x9cMxe2x80x9d are functions of time.
Detection and interpretation of the chlorophyll fluorescence intensity will be discussed now.
The detection of sun-induced chlorophyll fluorescence is difficult due to the fact that the fluorescence signal is superimposed by the reflected light (passive spectrum). For leaves or plant canopies the fluorescence signal is of the order of only some percent compared to the total signal. Therefore, different measuring techniques applying additional light sources were developed in the past for using the chlorophyll fluorescence for different applications.
In general, a modulated or pulsed light source is added to the sun irradiation xe2x80x9cIAbs-Sunxe2x80x9d inducing a modulated or pulsed fluorescence signal Fadd(t) which superimposes the sun induced fluorescence FSun(t) and the reflected signal IR(xcex). Applying a laser source for excitation the so called laser induced fluorescence (LIF) is generated. Equation (1) is then modified to:                     F        Sun            ⁢              (        t        )              +                  F        add            ⁢              (        t        )              =                    k        Fluorescence                              k          Fluorescence                +                              k            Photosynthesis                    ⁢                      (                          Φ              ,              M                        )                                *                  ∫        PAR            ⁢                        (                                    I                              Abs                -                Sun                                      +                          I              add                                )                ⁢                              ⅆ            λ                    .                    
The total signal which is normally detected is given by the sum of all fluorescence signals and the reflected signal IR(xcex). With adequate technical set-up the fluorescence signal excited by an additional light source can be separated from the passive spectrum and the sun induced fluorescence even under daylight conditions at distances, ranging from direct contact (Schreiber 1986, Patent DE 3518527, Mazzinghi 1991, EP 0 434 644 B1) to one meter (Chappelle 1995, U.S. Pat. No. 5,412,219) and several hundred meters (Cecchi and Pantani 1995, EP 0 419 425 B1).
The technical challenge for all systems either for contact measurements as well as for remote measurements is to install an excitation set-up strong enough to induce a sufficiently intense fluorescence signal in order to overcome the passive spectrum and weak enough to keep the photosynthetic system in an unchanged physiological status.
In the well known pulse-amplitude-modulation (PAM) fluorometer (Schreiber et al. 1986, Patent DE 3518527) a weak measuring light (light emitting red diode LED) induces the chlorophyll fluorescence via an optical fiber without changing the photosynthetic state of the plant. The fluorescence is transmitted by an optical fiber to a photodiode which detects all fluorescence light above 700 nm. For dark adapted plants no photosynthetic activity is stimulated when the measuring light is on.
Illumination of a dark adapted leaf with an intense flash of several milliseconds up to some seconds duration (called saturating light pulse) gives the maximum available fluorescence (called: Fm) but does not induce photosynthesis. A continuous illumination with non saturating light (called: actinic light) induces photosynthetic activity. After several seconds until minutes of illumination all contributing processes are in equilibrium with the supplied light and thus the fluorescence has reached a steady state value Fs. The transient of the fluorescence during illumination of dark adapted leaves is called Kautzky effect. For example FIG. 1 shows a measured diagram of a Kautzky kinetic of a cucumber plant. The detected fluorescence at 685 nm is exclusively induced by the laser pulses. Illumination with a 500 W halogen spot light influence the photosynthetic state only and thus kPhoto. Its contribution to the fluorescence signal, especially as excitation source, is negligible. The PAM fluorometer is normally operated in direct contact with leaves but can be used also at distances of some centimeters.
Detection and interpretation of the Red Fluorescence Ratio will be discussed now.
When excited by UV light, the typical fluorescence spectrum of a plant exhibits two dominant emission bands (FIG. 2), one from 400 nm-600 nm (called: blue-green fluorescence BG) and one from 650 nm-800 nm (called: red fluorescence; F685, F730). For example FIG. 2 shows a diagram of the fluorescence emission spectrum of a maize plant grown in the greenhouse. The fluorescence at 685 nm and at 730 nm (called: F685 and F730) originates exclusively from the leaf internal-chlorophyll. The blue-green fluorescence (BG) is emitted primarily by phenolic components of the cell walls.
The emission features of healthy plants are closely coupled to the plant morphology, as e.g. the pigment constituents and pigment concentration. Additional features may occur when plants are infected by fungi.
From experiments it is known, that the emissions at 685 and 730 nm are both linked to the photosynthetic system as described before and thus show nearly the same variation in time. In contrast the fluorescence ratio F685/F730 of an individual plant or leaf is constant in time and depends only on the optical properties of the leaf (Equation (2)).                               F685          F730                =                              Ψ            730            685                    ⁢          A          ⁢                      xe2x80x83                    ⁢                                                    ⅇ                                                      -                                          (                                                                        β                          ⁢                                                      xe2x80x83                                                    ⁢                          1                                                +                                                  c                          *                          α                          ⁢                                                      xe2x80x83                                                    ⁢                          1                                                                    )                                                        ⁢                  d                                            -              1                                                      ⅇ                                                      -                                          (                                                                        β                          ⁢                                                      xe2x80x83                                                    ⁢                          2                                                +                                                  c                          *                          α                          ⁢                                                      xe2x80x83                                                    ⁢                          2                                                                    )                                                        ⁢                  d                                            -              1                                                          (        2        )            
with:
"psgr":=spectral fluorescence characteristic @xcex=685 and 730 nm
xcex2:=scattering coefficient @xcex=685 and 730 nm
c:=chlorophyll concentration
xcex1:=specific absorption coefficient @xcex=685 and 730 nm
d:=leaf thickness
A:=constant, also containing the coefficients xcex1,xcex2,c,d.
The fluorescence emission and the pigment absorption bands are overlapping around 685 nm (FIG. 3), hence the emitted (fluorescence-) photons are reabsorbed selectively during their path through the leaf tissue resulting in an exponential dependence of the ratio F685/F730 from the parameter: mean free light path in the leaf, scattering coefficient and chlorophyll concentration. FIG. 3 shows a diagram of the shape of specific absorption (xcex1) of chlorophyll a and the corresponding fluorescence emission spectrum (xcexa8).
The only time dependent variation found in the ratio occurred during the transitions from fully dark adapted plants to light adaptation. This small variation was shown to occur from dark to early morning, from afternoon to evening and during Kautzky kinetic. Under day light no significant dependence or correlation respectively of the red fluorescence ratio and global irradiation could be found. Therefore it is assumed that these changes are related to variations in the optical properties of the leaf tissue. A potential mechanism could be the orientation of the plant organelles (e.g. chloroplasts) towards the arising illumination, but this is matter of further investigations.
Nevertheless, the ratio gives access to measure relative variations of the chlorophyll concentration for a plant species if one assumes a similar morphology for the individual plants. This means that the leaf internal scattering coefficient and the leaf geometry are comparable.
Mazzinghi (EP 0 434 644 B1, 1991 and P. Mazzinghi: xe2x80x9cA laser diode fluorometer for field measurements of the F685/F730 chlorophyll fluorescence ratioxe2x80x9d in xe2x80x9cREVIEW OF SCIENTIFIC INSTRUMENTSxe2x80x9d, Vol.67, No. 10, October 1996, pages 3737-3744, XP000635835, New York, USA) developed an xe2x80x9cinstrument for the two-channel measurements of the fluorescence of chlorophyllxe2x80x9d. This portable and compact system is dedicated for direct contact measurements of the fluorescence ratio F690/F730 (respectively F685/F730) as well as for measurements of the RFD-value at both wavelengths using a helium neon laser or a laser diode as continuous excitation source. When operated in full sunlight the residual background light (passive spectrum), due to the direct reflection of the leaf, must be checked after each measurement separately (and then subtracted) because this light is not completely eliminated by the filter on the probe.
The blue-green fluorescence BG will be discussed now.
The origin of the BG fluorescence is more difficult to identify and is still matter of scientific discussion. The blue-green fluorescence originates mainly from the cell walls in the upper layer of leaves and only a small fraction is emitted from deeper cell layers.
For chloroplasts no blue fluorescence is evident because the red chlorophyll fluorescence is the dominant factor. Nevertheless it is known that NADPH in the chloroplasts is emitting blue fluorescence. Also on the cell level it is shown that fluorescent co-enzymes such as NADH or NAD(P)H are very sensitive bio-indicators of metabolic functions such as the degradation of glucose or respiration. Thus the blue NADPH emission depends on the physiological state of the plant.
For leaves the emission of enzymes and co-enzymes is completely covered by emission of the cell wall where several pant constituents are embedded. As is well-known plant phenolics, ferulic-, chlorogenic- and caffeic acids, as well as coumarins are source of the blue emission and alkaloids and flavonols are source of the green fluorescence.
The detection and interpretation BG fluorescence intensity will be discussed now.
On the basis of the present knowledge about the BG fluorescence there is no commonly agreed interpretation of the overall BG fluorescence intensity. A lot of emitter are clearly identified but their contribution to the total signal is still unknown.
A link to the photosynthetic apparatus, comparable to the description in Equation (1), is only found for the NADPH fluorescence. Assuming a time invariant BG fluorescence of all other emission sources it could be capable to monitor also this transients of the BG fluorescence.
Generally the emission is originated at other plant components, e.g. the epidermal cell layer, especially the cell walls or at the vacuoles, also in the mesophyll cells. All this component do not contain chlorophyll and thus do not contribute to the photosynthesis. Nevertheless the main information, derivable from the BG fluorescence intensity is an estimation of the quantity of emitting plant (tissue) pigments in this spectral range.
Detection and interpretation of the parameter BG fluorescence ratio will be discussed now.
Evaluating the spectral characteristics of the BG fluorescence with special regard to the also monitored red fluorescence provides the possibility to normalize the fluorescence (to the chlorophyll fluorescence) and thus making this measurement resistant to calibration effects and signal fluctuations from successively recorded measurements.
As investigated there are at least four different effects, proved by comparable laboratory or field experiments, which can be differentiated due to the spectral characteristics in the blue, green and red range:
Distinction of mono- and dicotyledone plants (blue-green-red)
Synthesis of UV protection pigments (UV stress) (blue-red) Infection by mildew, rust, . . . (fungi) (blue-green-red)
Detection of leaf necrosis at pine needles (blue-green).
In the case of leaf surface coverage by other organic materials as e.g. during fungal infections the fluorescence emission spectrum of the infected leaf is affected in two different ways:
the auto-fluorescence of the fungi increases (or changes) the BG fluorescence selectively
fungi at the surface lower the red fluorescence by absorbing the excitation light and therefore decreasing the penetration depth. The same effect is seen if the excitation light is diffusely reflected by an additional tissue layer at the plant surface.
The latter behaviour is also known from UV protecting pigments within the epidermal cell vacuoles which hinders the xe2x80x9cUVxe2x80x9d excitation to penetrate deeper cell layers and thus depresses the chlorophyll fluorescence selectively. Usually these pigments (e.g. anthocyanin) are solely absorbers and do not contribute to the total fluorescence signal.
The following preconditions are to satisfy for a successful data collection.
Actinicxe2x80x94non actinic measurement light conditions are to be considered.
Depending of the topic of interest it may be necessary to avoid an influence to the photosystem by the excitation source. In all cases where the fluorescence intensity is relevant for the measurement the excitation must keep the plant system condition. It should be controlled only by environmental parameters as e.g. solar irradiance, vitality or the healthy state.
An a priori excluded influence of the excitation allows a measurement of the illumination and thus an estimation of the vitality or healthy state respectively.
This is mostly irrelevant for measurement of the relative chlorophyll concentration because both emission bands are dependent in the same way, but already in the case of comparing the red fluorescence with the blue fluorescence the different origins of the emission bands indicate the necessity of controlling the emission intensity as far as possible.
On the other hand an undisturbed (by excitation light) photosystem gives the possibility to extract plant specific information by controlling the environmental parameter. The variation of e.g. the light energy supplied or the health state of a plant allows the determination of measurement rules to make an interpretation of the intensity fluctuations feasible. This technique is widely used in the already mentioned PAM fluorometry, or realized by the daily cycle measurements with the far field lidar system.
Beyond that the signal to background ratio (SBR) is to be considered.
The signal to background ratio for the active induced fluorescence is defined as the number of photons passively reflected by the leaf tissue (IR) plus sun induced fluorescence (FSun) plus the emitted fluorescence photons (stimulated by the excitation of the measurement light (Fadd)) divided by the number of photons passively reflected by the leaf tissue (IR) plus sun induced fluorescence (FSun)
To distinguish each contribution one needs to determine both of them. In the far field lidar technique the excitation pulse is as intense that the passive background is negligible in comparison to the induced fluorescence light. The main disadvantages of this method are the high cost for an adequate excitation system (laser), the huge effort to operate the laser (power supply, eye safety restrictions, precision optics) and the uncertainty of the illumination state at the measurement plant position.
Moreover the signal to noise ratio (SNR) must be considered.
For single shot operation the signal to noise ratio defines whether a detection system is able to measure the fluorescence signal with each excitation pulse. The main source of noise defining the SNR are:
the sensitivity of the photon detector
the power of the background signal (St1)
the power of the active fluorescence signal (Flaser)
the operating distance
the entrance aperture of the detection system.
The first source is defined by the detector characteristics, whereas the other three components are dependent on the so called xe2x80x9cshot-noisexe2x80x9d.
Photo multiplier tubes (PMTs), especially in continuous operation, are detectors with extremely low noise levels, clearly below the noise level of the photon (shot) noise even with a high gain level. With an optical system large enough to collect sufficient fluorescence photons to depress the signal distortion by shot noise allows single shot operation. This is mandatory in all cases where the target is rapidly changing.
If the target is located at a fixed position relative to the detection system one may use for instance lock-in technique to separate a noisy fluorescence signal from any noise source independently whether the origin of the noise is shot- or detector noise. In this cases one can reduce the requirements to the optical system (reduction of the aperture) or exchange the photo multiplier tube by a cheaper avalanche or normal photodiode.
A fast repetition rate fluorometer is proposed in U.S. Pat. No. 5,426,306 for measuring in-vivo fluorescence of phytoplankton or higher plants with series of fast repetition rate excitation flashes. The system induces the variable fluorescence in order to derive photosynthetic parameters such as variable fluorescence, effective absorption cross section, rate of electron flow and turnover times of photosynthesis. This device is used for measuring fluorescence of samples as a function of the series of excitation flashes.
A method for the automatic detection of plants by measuring chlorophyll fluorescence intensity was introduced by WO 91/10352. According to this method, fluorescence is excited by a light source at wavelength under 550 nm. The emitted fluorescence is detected with a camera supplied with a broadband edge filter (transmission over 600 nm and blocking below 600 nm). No active background suppression is applied. Therefore, a recommendation is given that the light source is strong enough for picture information and that the radiation of the light source reflected directly from the plant or the substrate does not reach the camera.
It is an object of the invention to provide a cheaper high performance fluorescence detection assembly reducing the necessary excitation power by use of a low power laser sufficiently powerful to stimulate emission of a measurable quantity, and reducing the influence of the background signal.
It is a further object of the present invention to provide a new technical approach to measure well known plant physiological parameters under certain conditions with the most accurate determination of the corresponding measurement and environment conditions.
According to the present invention a fluorescence detection assembly for determination of relevant vegetation parameters is characterized in that said low power laser device provided in the excitation source is a high repetitive pulsed laser device with several nanosecond pulse length and a preferred excitation wavelength in the red spectral region of preferable 670 nm, in that said dichroic beam splitter couples the formed excitation beam co-axially to the optical axis of a receiver optic and directing this formed beam witout optical waveguiding to a vegetation target subject to be investigated, in that said basic fluorescence detector system forms an image of the excitation spot at the sensitive detector area, in that said electronic detection device operates at the doubled pulse repetition rate of said excitation laser source and samples the active fluorescence signal synchronously with the laser emission on the one hand and the passive background signal with a fixed delay in the microsecond range before or after the active signal on the other hand, recording those signals by means of a fast sample and hold circuit coupled to an analog to digital converter which enables a digital signal processing, in that said electronic detection device further comprises means for determining the pure fluorescence signal by subtracting the background signal from the active fluorescence signal electronically or in a post processing procedure, and in that said electronic trigger and timing device synchronizes the laser pulses with the sample intervals of said electronic detection device.
Thus this assembly measures explicitly the background signal. The interesting fluorescence signal Flaser is calculated by subtracting the passive contribution to the total signal.
Flaser=St2xe2x88x92St1
xe2x80x9cSxe2x80x9d is the signal at time (subscript) xe2x80x9ct1xe2x80x9d and xe2x80x9ct2xe2x80x9d
St1=IR+FxcexSun
St2=IR+FxcexSun+Fxcexlaser.
At xe2x80x9ct1xe2x80x9d the active excitation is zero and at xe2x80x9ct2xe2x80x9d the active fluorescence emission is added to the passive signal.
To reduce the necessary excitation power the detection spot and thus also the excitation spot is reduced as far as the contribution of the background signal is reduced to the level of the active fluorescence signal.
These and other features and advantages of the present invention will become more apparent from the following detailed description of exemplary embodiments thereof, as illustrated in the accompanying drawings, in which: